【作者】 范秀萍；

【导师】 陈洪岩；

【作者基本信息】 东北农业大学 ， 动物学， 2009， 硕士

【摘要】 猪传染性胃肠炎(Porcine transmissible gastroenteritis, TGE)是由猪传染性胃肠炎病毒(Porcine transmissible gastroenteritis virus, TGEV)引起的一种高接触性传染病,主要引起一周龄以下仔猪发生呕吐、水样腹泻和死亡(死亡率通常为100%)。猪传染性胃肠炎流行遍布全世界,给养猪业带来了巨大的损失。猪呼吸道冠状病毒(Porcine respiratory coronavirus, PRCV)是TGEV的基因缺失变异株,其与TGEV核苷酸序列同源性达96%。TGEV和PRCV基因结构十分相似,抗原相关,产生的中和抗体有交叉反应,传统血清学方法很难将二者区分开。PRCV与TGEV相比,其S基因5’末端有大量碱基缺失,不同PRCV毒株的碱基缺失情况各不相同,缺失从621～681个碱基不等,并导致PRCV失去两个抗原位点。目前,各国PRCV感染现象呈现日益严重的趋势,并且常在TGEV感染猪群中发生,造成TGEV血清转阳现象,因此建立一种TGEV与PRCV的鉴别诊断方法尤其重要。本研究根据已发表的PRCV基因组序列中的S基因序列和本实验室测定的TGEV华毒弱毒株的S基因序列进行比对分析,在S基因缺失区两端分别设计了两套引物P1、P2、P3、P4,以TGEV和PRCV细胞培养物为材料提取RNA,进行套式RT-PCR特异性片段扩增,TGEV P1、P2扩增片段大小为1338bp,P3、P4扩增片段大小为936bp,PRCV扩增片段分别为717bp、315bp。经过正交设计优化后建立的套式RT-PCR方法经过特异性、敏感性试验检测,只有TGEV与PRCV有特异性条带被扩增出,其他病毒(PEDV、PRRSV、SIV、PRoV、PRV、HCV)均未扩增出特异条带,TGEV与PRCV病毒最低检测量均为4 TCID50/mL,证明本法具有快速、特异和高度敏感的特点。本研究还设计建立了环介导等温扩增方法用来鉴别区分TGEV与PRCV。根据已发表的PRCV基因组序列中的S基因序列和本实验室测定的TGEV华毒弱毒株的S基因序列进行对比分析,在S基因缺失区和共同区分别设计一套引物Q-FIP、Q-BIP、Q-F3、Q-B3与QX-FIP、QX-BIP、QX-F3、QX-B3,以TGEV与PRCV细胞培养物为材料提取RNA,进行特异性LAMP片段扩增,其中TGEV在Q系列引物扩增下会出现典型的LAMP梯度条带,Q系列引物扩增最小带为184bp,而PRCV在QX系列引物扩增时出现条带,片段大小194bp。经过正交设计优化后建立的LAMP方法经过特异性、敏感性实验检测,只对TGEV与PRCV扩增出现条带,而其他病毒(PEDV、PRRSV、PRoV)无条带出现,均为阴性。此方法可检测到的TGEV与PRCV病毒最低量为0.1TCID50/mL。证明本法具有快速、特异和高度敏感的特点。本方法采用美莱博公司的荧光染料SmartGreenⅠ与MgCl2进行肉眼及荧光可视检测,可以很容易的区分出阴阳性反应,方便现地检测病料。上述两种方法的建立可以很好地鉴别诊断和区分TGEV与PRCV病毒,并且两种方法的特异性和敏感性都很高,相比较起来RT-LAMP方法比套式RT-PCR方法更敏感,且更适于现地病料检测。本研究采用原核表达的TGEV N蛋白免疫BALB/c小鼠,按常规方法进行细胞融合,间接ELISA方法进行筛选,抗原为TGEV N蛋白。采用有限稀释法,经过四次克隆,最终获得三株抗TGEV N蛋白的杂交瘤细胞株(1A9、1G7、1H8)。三株杂交瘤细胞分泌的单克隆抗体亚型均为IgG1型,杂交瘤细胞经过体外长期培养和冻存均未影响抗体的分泌。间接ELISA检测1A9、1G7、1H8细胞培养上清的抗体效价分别为1:640、1:320、1:800,腹水抗体效价分别为1:6400、1:6400、1:12800。用获得的单克隆抗体和SP2/0细胞培养上清为一抗,感染TGEV的ST细胞培养物为抗原进行间接免疫荧光实验,荧光二抗孵育作用后,与单克隆抗体作用的细胞培养物中,多数细胞在其胞浆和细胞膜上出现强荧光,与SP2/0细胞培养上清作用未见出现荧光,结果表明,利用所制备的单克隆抗体进行间接免疫荧光对病原检测具有较高的特异性。用单克隆抗体与TGEV和TGEV N蛋白进行westernblot实验:荧光素二抗作用后用红外荧光扫描成效系统进行扫描,发现只在TGEV N蛋白处出现特异性条带,而TGEV病毒其他蛋白处未有特异性条带出现。结果说明,利用所制备的单克隆抗体进行westernblot对病原检测具有较高的特异性。上述单克隆抗体的制备为下一步鉴别诊断TGEV/PRCV的阻断ELISA方法的建立打下了前期基础,为TGEV/PRCV的血清学诊断提供了材料。更多还原

【Abstract】 Porcine transmissible gastroenteritis virus (TGEV) is the causative agent of transmissible gastroenteritis of swine, which causes piglets vomit, diarrhea and death (100% high mortality). It is a world disease and causes great harm to swine industry. Porcine respiratory coronavirus (PRCV) is a natural deletion mutant of TGEV, whose homoly of nucleotide sequence reaches 96% compared to TGEV. Compared to TGEV, there are 621～681 nucleotides deletion at the N-terminal of S gene of PRCV, making PRCV lose two antigen sites. Because of similar antigenicity and morphology, TGEV couldn’t be differentiated from PRCV by traditionary serodiagnosis and electron microscope. Today,more and more swine herds infect PRCV all over the world. So that, it is very necessary to constrcuct a quick and sensitive detection method.Thus, we developed a nested RT-PCR method targeted to the deletion in Spike gene of PRCV to differentiate and diagnose TGEV from PRCV. Two pairs of primers, P1 and P2, P3 and P4, were designed based on S gene nucleotide sequences of TGEV and PRCV, after sequences compared between PRCV published and TGEV attenuated-H strain. Nested RT-PCR was developed to amplify specific sequences, after extracting RNA from TGEV and PRCV cell cultures. The amplified segment with P1 and P2 of TGEV gene was 1336bp, P3 and P4 936bp and that of PRCV gene was 717bp, 315bp. This method was optimized by Orthogonal design. Specific straps were amplificated with PRCV and TGEV as templates, but not with other viruses (PEDV、PRoV、PRRSV、PRV、HCV、SIV). And the lowest quantity of both TGEV and PRCV were 4 TCID50/mL. Results showed that the nested PCR was rapid, specific and sensitive in detecting the clinical samples.We developed a loop-mediated isothermal amplification(LAMP) method to differential diagnosis TGEV and PRCV. Comparing the S protein sequences of PRCV published and TGEV attenuated H strain, we designed a pair of primers , named Q-FIP, Q-BIP, Q-F3, Q-B3 and QX-FIP, QX-BIP, QX-F3, QX-B3, targeted different and the same sequence on S gene respectively. LAMP was developed to amplify specific sequences, after RNA extracted from TGEV and PRCV cell cultures. There were a typical ladder strap using TGEV RNA as template, and the smallest amplificated straps were 184bp. And there was a typical ladder strap using PRCV RNA as template with QX-primers, and the smallest strap was 194bp. The amplified products were analyzed by agarose gel electrophoresis or visualized with SmartGreenⅠand MgCl2 as stain. This method was optimized by Orthogonal design. The results demonstrated that the RT-LAMP assay had no cross- reaction with other swine viruses (PEDV, PRRSV, PRoV). And the lowest quantity of both TGEV and PRCV were 0.1TCID50/mL. Therefore, the RT-LAMP assay provides a specific and sensitive means for detecting TGEV and PRCV in a simple, fast, and costeffective manner. And the visualized analysis could help us tell positive products from the negative.Both nested RT-PCR and LAMP are effective to differential diagnosis TGEV and PRCV specifically and sensitively. Furthermore, the RT-LAMP assay is more sensitive and can be performed in less well- equipped laboratories as well as fields.In this research, recombinant neucleocapsid protein of transmissible gastroenteritis virus(TGEV) which was expressed with prokaryotic expression system was used to immune BALB/c mice. Cell fusion was performed previously. Indirect ELISA assay was carried out to screen hybridoma cell lines using TGEV N potein as the antigen. By limiting dilution and 4 serial of clones, 3 hybridoma cell lines(named 1A9、1G7、1H8, respectively) secreting antbodies against TGEV N protein were obtained. The antibodies of all the three hybridoma cell lines belong to IgG1 subgroup. After extended culture and several freeze-thaw cycles, the monoclonal antibodies wre still stably secreted. The average number of chromosomes in 3 hybridomas was 84～100. Investigated through indirect ELISA, the titers of 3 cell-cultured antibodies were measured to be 1:640、1:320、1:800, and the titers of antbodiees produced in ascities were 1:6400、1:6400、1:12800, respectively. Immunofluorescence assays were performed with ST cell monolayers infected with TGEV probed by the antibodies or supernatant of SP2/0 cell culture.After staining with FITC-labeled secondary antbodies, strong yellow-green fluorescence was observed in plasma and on the membrane of cells. However, no fluorescence was detected with the addition of supernatant of SP2/0 cell culture. With recombinant N protein as immunogen, antibodies were found to specifically bind to pathogens with the confirmation of indirect immunofluorescence assays. Using purified TGEV and TGEV N protein as antigen, westernblot was performed to determine the correlation between the monoclonal antbodies and TGEV/TGEV N protein. After staining with fluorescein-labeled secondary antbodies, it was revealed that the supernatants of 3 hybridomas but not supernatant of SP2/0 cell culture recognized TGEV and TGEV N protein. The specificity of antibodies to virus was further ensured.The monoclonal antibodies against TGEV N protein could provide foundation for the development of blocking-ELISA of differential diagnosis TGEV and PRCV.更多还原